Eukaryotic DNA is packaged into highly ordered structures knows as chromatin that further packs the DNA into higher-order structures, limiting the accessibility of the underlying genetic information for the processes like transcription, replication, and repair. However, eukaryotic cells have evolved proteins called chromatin regulators that regulate the accessibility of the genetic information when needed. This dissertation aims to characterize the role of two such proteins, JARID2 and the polycomb repressive complex-2 (PRC2), during skeletal muscle proliferation and differentiation.JARID2 is an inactive yet evolutionarily conserved histone demethylase that is shown to be a sub-stoichiometric component of the PRC2 complex. The PRC2 complex represses gene expression through the trimethylation of lysine 27 of histone 3 (H3K27me) tails. H3K27 methylation leads to chromatin compaction. JARID2 helps in targeting of the PRC2 complex to its target loci. JARID2 is shown to be required for the normal development of mice, as loss of Jarid2 leads to lethality in utero. We, for the first time, show that JARID2 is required for the normal skeletal muscle differentiation. We show that the JARID2 regulates the expression of myogenic regulatory factor, Myod1, both through direct repression and activation through the modulation of canonical Wnt signaling pathway. JARID2, in association with the PRC2 complex, represses Wnt antagonist Sfrp1 to modulate the activity of the canonical Wnt signaling pathway. The translocation of Wnt effector protein, b-catenin, from the cytoplasm to the nucleus modulates the activity of the canonical Wnt signaling pathway during activation. We also show that b-catenin directly regulates the expression of Myod1 gene through its direct binding in the distal regulatory region.We further extend the role of JARID2 during skeletal muscle proliferation. We show that JARID2 also plays an essential role in restraining the skeletal muscle proliferation through its direct repression of positive cell cycle regulators cyclin D1 (Ccnd1) and cyclin E1 (Ccne1). Furthermore, we show that retinoblastoma protein 1 (Rb1), a negative regulator of cell proliferation that promotes cell cycle exit and differentiation, is also directly regulated by JARID2 in PRC2 dependent manner. Together, we show that JARID2 precisely controls cell proliferation and differentiation during skeletal muscle differentiation.Further, we show that the regulation of cell proliferation by JARID2 is PRC2 complex dependent. When the PRC2 complex was depleted or inhibited to a modest level, the cells have an increased cell proliferation ability compared to severe loss or inhibition of EZH2, the catalytic subunit of the PRC2 complex, that leads to the apoptosis of the cells. It is also marked by increased expression of known PRC2 targets genes. We show that the increased proliferation upon modest inhibition or depletion of EZH2 is through direct de-repression of positive cell cycle genes, Ccnd1, and Ccne1. It is the first work that shows a context-dependent role of the PRC2 complex during skeletal muscle proliferation and differentiation.My dissertation also makes an extraordinary discovery as to why myogenin is required for the proper function of MyoD during skeletal muscle differentiation, even though both proteins share a large set of overlapping target genes. We show that myogenin is required for the nucleosome disassembly and reassembly at the target genes through recruitment of the FACT complex, a histone chaperone. We also show that myogenin is required for the assembly of the basic transcription machinery and RNA polymerase II to the target muscle genes during differentiation. Surprisingly, we also show that myogenin reinforces its own expression through the activation of Myod1 expression during skeletal muscle differentiation. Myogenin is a known target of MyoD.Taken together, this dissertation provides a molecular mechanism for the crosstalk between a signaling pathway with chromatin regulatory proteins, JARID2, and the PRC2 complex in regulating skeletal muscle differentiation. It also extends the role of JARID2 and the PRC2 complex - known oncogenes, in precise, context-dependent control of cell proliferation and differentiation in skeletal muscle.

Skeletal muscle is a highly adaptable tissue that comprises approximately 40% of total body weight while accounting for up to 90% of whole-body oxygen consumption and energy expenditure during exercise. The loss of skeletal muscle protein and subsequent decrease in muscle mass (atrophy) that accompanies disuse results primarily from a decrease in intracellular protein synthesis combined with an increase in proteolytic activity. Interestingly, these processes of skeletal muscle atrophy are amplified by changes in mitochondrial capacity, with evidence suggesting that the maintenance of mitochondria during periods of disuse protects skeletal muscle against atrophy. Remarkably, rodents with denervated muscle are protected against muscle atrophy following whole-body heat stress. The mechanism of protection appears to be tied to the observed increases in heat shock protein (HSP) and PGC-1α, which accompany the heat stress. Without any published observations as to whether such heat-induced protection against muscle atrophy would translate to human muscle, the aim of this project was to determine the extent to which deep tissue heating (via pulsed shortwave diathermy) might provide protection against skeletal muscle atrophy.

Purpose: To investigate the effects of eccentric exercise on lower body skeletal muscle mass during rapid body mass loss induced by bariatric surgery. Methods: All participants began 6 to 8 weeks after undergoing Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG). Skeletal muscle mass (SMM) in the lower body was measured via magnetic resonance imaging (MRI); additional exercise measurements included muscular strength and functional capacity. Quality of life was measured using Short Form 36 (SF-36). Nineteen females (age = 37.6 ± 9.8 yr, height = 164.4 ± 7.2 cm, mass = 106.9 ± 15.6 kg) were randomly assigned to 1 of 3 groups: eccentric exercise (EEX; n = 6), concentric exercise (CEX; n = 7), or standard-of-care control (CON; n = 6). Exercise groups performed 30-minute lower-body exercise sessions 3 times per week for 16 weeks. Each month the exercise tests were evaluated. At the end of 16 weeks, all participants performed the final exercise tests, received a final MRI scan, and completed the SF-36 questionnaire. Results: Thirteen individuals completed the study. All groups lost mass: CON: 21.4 ± 3.7 kg (p < 0.001), CEX: 19.9 ± 4.0 kg (p = 0.001), and EEX: 21.8 ± 3.3 kg (p < 0.001). SMM decreased in all groups: CON: 0.77 ± 0.5 kg (p = 0.18), CEX: 1.19 ± 0.6 kg (p = 0.06), and EEX: 0.90 ± 0.5 kg (p = 0.09). The skeletal muscle loss in percent of total mass loss was 3.7 ± 4.1%. All measures of muscular strength showed no difference, except for a small decrease in dynamic (60°·sec-1) strength in the eccentric group. Functional capacity and physical quality of life increased significantly in all groups (p < 0.05). Conclusion: SMM loss still occurred in the lower body regardless of resistance training, but the loss was less than what was previously documented. Improved postsurgical functional capacity and physical quality of life may be due to a reduction in fat mass and maintenance of muscular strength during the period of rapid mass loss.

The extracellular matrix (ECM) of the skeletal muscle provides the framework for the muscle structure and plays a key role in the repair and maintenance of myofibers through the resident fibroblasts and muscle satellite cells. However, excessive production of ECM components, notably collagen, leads to fibrosis which impedes muscle function, impairs the natural repair process, and leads to muscle weakness. Fibrosis is a hallmark of muscular dystrophies, including Duchenne muscular dystrophy (DMD). Duchenne muscular dystrophy is a terminal, x-linked disorder characterized by progressive muscle wasting as muscle fibers are replaced by fibrosis and fat. There are approximately 300,000 DMD patients worldwide, and the few disease modifying treatments are genotype specific, only helping a small percentage of the patient population. Myostatin is a member of the transforming growth factor beta (TGF-β) family of ligands, is a negative regulator of muscle mass, and may also contribute to the fibrotic environment in dystrophic muscle through myofibroblast proliferation and survival. Therefore, myostatin blockade could potentially ameliorate muscle weakness in DMD patients by increasing skeletal mass and function while also reducing the accumulation of fibrosis. A murine anti-myostatin antibody, mRK35, and its humanized analogue, domagrozumab, are specific and potent inhibitors of myostatin. mRK35 was tested in multiple mouse models, from healthy C57Bl/6 and C57Bl/10, mildly dystrophic C57Bl/10.mdx, and severely dystrophic D2.mdx mice, for changes in muscle mass, muscle function, and fibrotic content. Additionally, inflammatory, fibrotic, and myogenic gene expression changes were analyzed in the severely dystrophic animals treated with mRK35. Domagrozumab was tested in non-human primates (NHPs) for changes in skeletal muscle mass. Myostatin blockade with mRK35 resulted in muscle anabolic and functional improvements in healthy murine models and NHPs treated with domagrozumab demonstrated a dose-dependent increase in lean mass and muscle volume. However, as mice age or as the dystrophic severity of the model increases, the anabolic effect of myostatin inhibition is diminished. The extensor digitorum longus (EDL) muscle escapes this trend and is the most responsive to myostatin inhibition across all mouse strains and disease severities. However, analysis of the fibrotic content in the triceps and diaphragms of D2.mdx mice treated with mRK35 for 8 weeks does not reveal any change in fibrotic content. Gene expression changes in the muscles within these mice appear to be tightly tied to their healthy or dystrophic state and myostatin inhibition has minimal effect. In sum, while specific myostatin inhibition with mRK35 increases muscle weight and function in mice, there is no conclusive evidence of reduced muscle fibrosis. / 2023-06-22T00:00:00Z

RNA interference (RNAi) is an important pathway that regulates gene expression in several organisms. The role of RNAi in post-transcriptional gene silencing in the cytoplasm is well characterized. In contrast, the role of RNAi components in the nucleus remains to be elucidated. Previous reports have indicated that RNAi components (Dicer and Argonaute proteins) and small RNAs act in the nucleus to regulate various pathways including heterochromatin formation, transposable elements repression, RNA Pol II processivity and alternative splicing. Nuclear Ago1 and Dicer have also been found to associate with active promoters and enhancers in mammalian cells, however their functional roles and mechanisms remain elusive. In this work, I investigated the functional role of nuclear RNAi components in gene expression regulation during skeletal muscle differentiation. To address this question, I undertook genomic and biochemical approaches applied to myogenic cells (C2C12) as a model system. I found that Ago1 and Dicer are present in the nucleus of C2C12 cells and expressed during differentiation. Chromatin Immunoprecipitation (ChIP) coupled with high throughput sequencing and quantitative real-time PCR indicate that Ago1 and Dicer are enriched at promoters and enhancer regions of myogenic genes. Interestingly, I found that depletion of Ago1 and Dicer reduces enhancer RNAs (eRNAs) levels at enhancer regions and expression of MyoD during differentiation. I observed that loss of Ago1 impacts differentiation, whereas, loss of Dicer leads to cell death and has severe effects on C2C12 cells. Moreover, using Chromosome Conformation Capture (3C), I revealed that Ago1 is involved in enhancer-promoter interaction at MyoD locus. The knockdown of Ago1 destabilizes these interactions and decreases the expression of MyoD. Finally, I demonstrated that Ago1 binds to eRNAs and interacts with CBP Acetyl-transferase in the nucleus of myotube cells. Ago1 depletion leads to loss of eRNA-CBP interaction and consequent impairment of CBP acetyltransferase activity and failure of MyoD mediated activation of the myogenic program. Taken together, these finding indicate that nuclear Ago1 together with eRNAs and CBP regulates MyoD expression by stimulating histone acetylation during differentiation. This study uncovered a novel function of chromatin associated Ago1 in gene expression regulation during mammalian skeletal muscle differentiation.

Caspase-3 has demonstrated a non-apoptotic function in several developmental programs including skeletal muscle differentiation, yet the mechanism of action has not been fully elucidated. Under apoptotic conditions Caspase-3 induces DNA fragmentation through activation of CAD. Recent observations have demonstrated CAD activity and the resulting DNA strand breaks are also vital for skeletal muscle differentiation. These breaks are transient in nature, suggesting an active DNA repair program to maintain genomic integrity. The aim of this study was to delineate the DNA repair mechanism coordinated with caspase/CAD mediated DNA damage. It was found that XRCC1 formed punctate nuclear foci early in myoblast differentiation concurrent to the induction of DNA damage. Caspase-3 inhibition caused attenuation of the formation of DNA lesions and XRCC1 foci in differentiating myoblasts. Targeted reduction in XRCC1 expression impaired myoblast differentiation. These results suggest that XRCC1 may play a role in repairing the DNA damage associated with myoblast differentiation.

No description available.

Skeletal muscle satellite cells (myoblasts) are the primary stem cells of skeletal muscle which contribute to growth, maintenance, and repair of the muscles. Satellite cells are the first stem cells used for cellular cardiomyoplasty more than 20 years ago. The isolation, culture, labeling, and identification of satellite cells are described in detail here. The implantation and outcomes of cellular cardiomyoplasty using satellite cells have been summarized in the previous chapter (Chapter 1).

Skeletal muscle satellite cells (myoblasts) are the primary stem cells of skeletal muscle which contribute to growth, maintenance, and repair of the muscles. Satellite cells are the first stem cells used for cellular cardiomyoplasty more than 20 years ago. The isolation, culture, labeling, and identification of satellite cells are described in detail here. The implantation and outcomes of cellular cardiomyoplasty using satellite cells have been summarized in the previous chapter (Chapter 1).

Skeletal muscle metastasis from breast carcinoma is a relatively rare clinical entity. We report two cases of breast cancer metastatic to the skeletal muscle, diagnosed by ultrasound guided fine needle aspiration (US-FNA) biopsy done by interventional cytopathologists at an outpatient cytopathology center. Our two patients presented with lower anterior neck firmness and chest wall mass, respectively. Ultrasound evaluation of our first case demonstrated hypo-echoic thickened anterior strap muscles while in the second case there was significant distortion of the anatomy from previous surgeries. It was necessary to proceed with FNA biopsy even when their ultrasound findings were equivocal, to establish a definite rapid diagnosis. The immediate onsite evaluation findings were suggestive of malignancy in both cases with subsequent core biopsy confirming the diagnosis of metastatic breast carcinoma. In cytopathology, point-of-care (POC) ultrasound is used as an adjunct tool that offers visual guidance during FNA of nonpalpable masses and enables sampling of lesional “hot” spots to ensure specimen adequacy. Studies have demonstrated a reduction in FNA nondiagnostic rates with the use of ultrasound-guidance consequently reducing health care costs associated with nondiagnostic FNAs. US-FNA also provides adequate samples for cell block preparations. Metastatic lobular carcinoma of the breast has a wide range of clinical presentations and a high level of suspicion is advised. Cytopathologists-performed US-FNA is a proven, less-invasive, cost-effective tool that provides timely cytologic diagnosis.